Fusion proteins, cells, and kits for characterizing Botulinum toxin

ABSTRACT

Compositions for characterization of Botulinum toxin (BoNT) are described that include a genetically modified cell that is transfected with an artificial construct comprising a nucleic acid sequence that encodes for a hybrid protein having (a) a reporter-containing portion chemically coupled to (b) a cleavage site and (c) a control fluorophore. The cleavage site interacts with a BoNT in a manner that cleaves the reporter-containing portion from remainder of the construct. The cleaved portion is destroyed or otherwise degraded by the local environment, and presence of BoNT is evidenced by reduction in signal from the reporter. The cleavage sequence is all or part of a SNARE protein, the cleavable reporter-containing portion is preferably Yellow Fluorescent Protein (YFP), Citrine, Venus, or a YPet protein and the control fluorophore is preferably CFP, mStrawberry, or a mCherry protein.

This application is a continuation of U.S. Pat. No. 9,274,121, filed onMay 31, 2012, which claims priority to U.S. Provisional Application No.61/492237, filed Jun. 1, 2011. The priority application, along with allother publications referenced herein are incorporated by reference tothe same extent as if each individual publication or patent applicationwere specifically and individually indicated to be incorporated byreference. Where a definition or use of a term in an incorporatedreference is inconsistent or contrary to the definition of that termprovided herein, the definition of that term provided herein applies andthe definition of that term in the reference does not apply.

FIELD OF THE INVENTION

The field of the invention is protease assays, especially those relatedto Botulinum toxins.

BACKGROUND

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum, andare among the most potent toxins known. These toxins are awell-recognized source of food poisoning, often resulting in seriousharm or even death of the victims. There are seven structurally relatedbotulinum neurotoxins or serotypes (BoNT/A-G), each of which is composedof a heavy chain ({tilde over ( )}100 KD) and a light chain ({tilde over( )}50 KD). The heavy chain mediates toxin entry into a target cellthrough receptor-mediated endocytosis. Once internalized, the lightchain is translocated from the endosomal vesicle lumen into the cytosol,and acts as a zinc-dependent protease to cleave proteins that mediatevesicle-target membrane fusion (“substrate proteins”).

These BoNT substrate proteins include plasma membrane protein syntaxin,peripheral membrane protein SNAP-25, and a vesicle membrane proteinsynaptobrevin (Syb). These proteins are collectively referred to as theSNARE (soluble N-ethylmaleimide-sensitive factor attachment proteinreceptor) proteins. Cleavage of SNARE proteins blocks vesicle fusionwith plasma membrane and abolishes neurotransmitter release atneuromuscular junction. Among the SNARE proteins, syntaxin and SNAP-25usually reside on the target membrane and are thus referred to ast-SNAREs, while synaptobrevin is found exclusively with synapticvesicles within the synapse and is called v-SNARE. Together, these threeproteins form a complex that is thought to be the minimal machinery tomediate the fusion between vesicle membrane and plasma membrane. BoNT/A,E, and C¹ cleave SNAP-25, BoNT/B, D, F, G cleave synaptobrevin (Syb), atsingle but different sites. BoNT/C also cleaves syntaxin in addition toSNAP-25.

Unless the context dictates the contrary, all ranges set forth hereinshould be interpreted as being inclusive of their endpoints andopen-ended ranges should be interpreted to include commerciallypractical values. Similarly, all lists of values should be considered asinclusive of intermediate values unless the context indicates thecontrary.

Due to their threat as a source of food poisoning, and as bioterrorismweapons, there is a need to sensitively and speedily detect BoNTs.Currently, the most sensitive method to detect toxins is to performtoxicity assay in mice. This method requires large numbers of mice, istime-consuming, and cannot be used to study toxin catalytic kinetics. Anumber of amplified immunoassay systems based on using antibodiesagainst toxins have also been developed, but most such systems requirecomplicated and expensive amplification process, and cannot be used tostudy toxin catalytic activity either. Although HPLC and immunoassay canbe used to detect cleaved substrate molecules and measure enzymaticactivities of these toxins, those methods are generally time-consumingand complicated, some of them require specialized antibodies, makingthem inapplicable for large-scale screening. Therefore, there is a needfor new and improved methods and compositions for detecting BoNTs.

In the last few years, researchers have started investigating use ofFRET assays to detect BoNTs. In FRET assays, two fluorigenic amino acidderivatives are used to replace two native amino acids in a shortsynthetic peptide (12-35 amino acids) that contain toxin cleavage sites.The fluorescence signal of one amino acid derivative is quenched byanother amino acid derivative when they are close to each other in thepeptide. This mechanism is called “Förster resonance energy transfer”(FRET). Cleavage of the peptide separates the two amino acidderivatives, such that a decrease in FRET can be detected.

FRET assays have been successfully used for detecting BoNTs. (See e.g.,U.S. Pat. App. No. 2004/0191887 to Chapman, filed Oct. 28, 2003, U.S.Pat. App. No. 2006/0134722 to Chapman, filed Dec. 20, 2004, U.S. Pat.No. 7,208,285 to Steward (April 2007), U.S. Pat. No. 7,183,066 toFernandez-Salas (February 2007), and application US2011/0033866 (publ.Feb 2010),

Although some success has been demonstrated in applying FRET assays todetection of BoNTs, the sensitivity and specificity are stillundesirable for many purposes.

In FRET assays for the localization of BoNT substrate proteins, forexample, measurements relative to the loss of FRET emission uponcleavage of the peptide can suffer from severe interferences, such thatin some cases there is no difference between cells treated with no BoNTversus cells treated with saturating concentrations of BoNT. Therefore,it can be said that methods based on the loss of FRET report BoNTinduced changes very poorly and thus its low statistical performance andreproducibility render it a non-reliable methodology. The US application2009/0191583 to Ester Fernandez-Salas claims a non-FRET BoNT assay usingonly a single fluorophore. The assays disclosed in this document usecells which are capable of efficient Clostridial toxin uptake and whichinclude a membrane localized Clostridial toxin substrate containing afluorescent marker.

As an example, a cell useful in this paper can express aSNAP25₂₀₆-enhanced green fluorescent protein (EGFP) fusion protein thatlocalizes to the plasma membrane. Upon BoNT/A treatment of this cell,cleavage of the membrane localized SNAP25₂₀₆-EGFP substrate occurs,releasing the EGFP containing fragment into the cytoplasm. Uponexcitation of the treated cell with a 484 nM laser, the EGFP is excitedand emits light at 510 nM. However, because a portion of the EGFP is nowcytoplasmic, a distribution change between the uncleaved, membranelocalized SNAP25₂₀₆-EGFP toxin substrate and the cleaved, cytoplasmiclocalized EGFP fragment can be observed in BoNT/A treated cells. Theassay might therefore work for qualitative analysis, but the presence ofEGFP emitting portions in both the membrane and the cytoplasm rendersthis method undesirable for quantitative analysis.

Thus, improved apparatus, systems and methods are therefore still neededwhich overcome the drawbacks and the limits of the prior art relative toboth FRET and non-FRET assays.

SUMMARY OF THE INVENTION

The inventive subject matter relates to a composition for testingpresence of an investigative substance , comprising a geneticallymodified cell having an artificial construct containing a nucleotidesequence that encodes for a hybrid protein having (a) areporter-containing portion chemically coupled to (b) a cleavage sitethat interacts with an investigational substance in a manner thatcleaves the reporter-containing portion from a remainder of theconstruct and (c) a control fluorophore selected and positioned in amanner such that, an increased concentration of investigationalsubstance does not lead to a coincidental increase in fluorescentemission of the control fluorophore.

As used herein, and unless the context dictates otherwise, the term“coupled to” is intended to include both direct coupling (in which twoelements that are coupled to each other contact each other) and indirectcoupling (in which at least one additional element is located betweenthe two elements). Therefore, the terms “coupled to” and “coupled with”are used synonymously.

In preferred embodiments, the investigational substance is a Botulinumtoxin (BoNT), and the cleavage sequence is appropriately matched withthe investigational substance. For example, the BoNT/A, E, and C cleaveSNAP-25, and BoNT/B, D, F, G cleave synaptobrevin (Syb), at single butdifferent sites. BoNT/C also cleaves syntaxin in addition to SNAP-25.

Contemplated cleavage site sequences can advantageously comprise (a) aSNARE protein, motif, or mutein. “Muteins” of a protein should beinterpreted herein as having at least 30% identity with a correspondingnative protein, including for example compositions having at least 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%identity with the native protein. Variations from identity can compriseany or more of additions, deletions and substitutions. Contemplatedmuteins include fragments, truncates and fusion proteins.

In other aspects of preferred embodiments, the cleavablereporter-containing portion comprises a fluorescent protein, as forexample, Yellow Fluorescent Protein (YFP). YFP is a genetic mutant ofgreen fluorescent protein, derived from Aequorea Victoria, and has anexcitation peak at 514 nm and emission peak at 527 nm.

Also contemplated for use in the cleavable reporter-containing portionare the closely related Citrine, Venus, and YPet proteins. Themodifications have reduced chloride sensitivity, faster maturation, andincreased brightness (product of the extinction coefficient and quantumyield). Of course, any of the fluorescent proteins mentioned herein canbe modified to include specific characteristics (e.g., spectral) or betruncated to a specific size.

Upon cleavage, the construct is cleaved into two parts, a reportercontaining portion that is destroyed or otherwise degraded by thecytosol or other local environment, and a second portion. To normalizethe signal detection, that second portion can advantageously include acontrol fluorophore, preferably at an opposite end from the reporter,which can assist in normalizing the assay. The control fluorophore can,for example, be Cyan Fluorescent Protein (CFP), mCherry, or mStrawberry.As a result of the position of control fluorophore in the construct, anincrease in the concentration of BoNT does not lead to a coincidentalincrease in fluorescent emission of the control fluorophore.

Hybrid protein(s) that are formed in the transfected cells preferablyinclude a transmembrane domain, which tends to locate to intracellularvesicles, and thereby present a vesicle-bound substrate. Heavychain-mediated endocytosis of the BoNT into the transfected cell isfollowed by presentation of the light chain on the outer surface of thevesicle, allowing the protease activity of the light chain to cleave thecleavage sequence of the hybrid protein(s), thus cleaving thereporter-containing portion, which then is destroyed or degraded toreduce the signal being tested. Full-length Syb, for example, contains116 amino acids, and is localized to vesicles through a singletransmembrane domain. In some contemplated assays, themembrane-anchoring domain comprises a fragment that contains apalmitoylation site. Suitable membrane-anchoring domains are described,for example, in US 20060134722 to Chapman.

While it is especially preferred that the transmembrane domain is thetransmembrane domain of synaptobrevin, mutations (e.g., transitions,transversions, insertions, deletions, inversions, etc.) thereof, andeven non- synaptobrevin transmembrane domains are also deemed suitablefor use herein. Similarly, it should be appreciated that thetransmembrane domain may also be replaced by another polypeptide moietythat allows at least temporary anchoring of the hybrid protein to amembrane such that the remainder of the hybrid protein is exposed to thecytosol.

With respect to the transfected cells expressing the hybrid protein itis generally preferred that the cell is stably transfected.Nevertheless, transient transfection is also contemplated. It is stillfurther typically preferred that the transfected cell is a neuronalcell. However, numerous other non-neuronal cells (including mammalian,rodent, and insect cells and even yeast and bacterial cells) are alsocontemplated herein. Most typically, the cells will constitutivelyexpress the hybrid protein(s) are therefore under appropriate regulatoryelements. In alternative aspects, the expression may also be induced.

According to a preferred embodiment, a recombinant nucleic acidmolecule, preferably an expression vector, encoding a BoNT substratepolypeptide and a suitable reporter is introduced into a suitable hostcell. An ordinarily skilled person can choose a suitable expressionvector, preferably a mammalian expression vector, and will recognizethat there are enormous numbers of choices. For example, the pcDNAseries of vectors, such as pCI and pSi (from Promega, Madison, Wis.),CDM8, pCeo4. Many of these vectors use viral promoters. Preferably,inducible promoters are used, such as the tet-off and tet-on vectorsfrom Clontech (Mountain View, Calif.).

Many choices of cell lines are suitable as the host cell. Preferably,the cell is of a type in which the respective BoNT exhibits its toxicactivities. In other words, the cells preferably display suitable cellsurface receptors, or otherwise allow the toxin to be translocated intothe cell sufficiently efficiently, and allow the toxin to cleave thesuitable substrate polypeptide. Specific examples include primarycultured neurons (cortical neuron, hippocampal neuron, spinal cord motorneuron, etc); PC12 cells or derived PC12 cell lines; primary culturedchromaffin cells; several cultured neuroblastoma cell lines, such asmurine cholinergic Neuro 2a cell line, human adrenergic SK-N-SH cellline, and NS-26 cell line. See e.g. Foster and Stringer (1999), GeneticRegulatory Elements Introduced Into Neural Stem and Progenitor CellPopulations, Brain Pathology 9: 547-567.

The coding region for the reporter/cleavage site construct is under thecontrol of a suitable promoter. Depending on the types of host cellsused, many suitable promoters are known and readily available in theart. Such promoters can be inducible or constitutive. A constitutivepromoter may be selected to direct the expression of the desiredpolypeptide. Such an expression construct may provide additionaladvantages since it circumvents the need to culture the expression hostson a medium containing an inducing substrate. Examples of suitablepromoters would be LTR, SV40 and CMV in mammalian systems; E. coli lacor trp in bacterial systems; baculovirus polyhedron promoter (polh) ininsect systems and other promoters that are known to control expressionin eukaryotic and prokaryotic cells or their viruses. Examples of strongconstitutive and/or inducible promoters which are preferred for use infungal expression hosts are those which are obtainable from the fungalgenes for xylanase (xlnA), phytase, ATP-synthetase, subunit 9 (oliC),triose phosphate isomerase (tpi), alcohol dehydrogenase (AdhA),.alpha.-amylase (amy), amyloglucosidase (AG—from the glaA gene),acetamidase (amdS) and glyceraldehyde-3-phosphate dehydrogenase (gpd)promoters. Examples of strong yeast promoters are those obtainable fromthe genes for alcohol dehydrogenase, lactase, 3-phosphoglycerate kinaseand triosephosphate isomerase. Examples of strong bacterial promotersinclude SPO₂ promoters as well as promoters from extracellular proteasegenes.

Hybrid promoters may also be used to improve inducible regulation of theexpression construct. The promoter can additionally include features toensure or to increase expression in a suitable host. For example, thefeatures can be conserved regions such as a Pribnow Box or a TATA box.The promoter may even contain other sequences to affect (such as tomaintain, enhance or decrease) the levels of expression of thenucleotide sequence. For example, suitable other sequences include theShl-intron or an ADH intron. Other sequences include inducibleelements—such as temperature, chemical, light or stress inducibleelements. Also, suitable elements to enhance transcription ortranslation may be present. An example of the latter element is the TMV5′ signal sequence (see Sleat, 1987, Gene 217: 217-225; and Dawson,1993, Plant Mol. Biol. 23:97).

The expression vector may also contain sequences which act on thepromoter to amplify expression. For example, the SV40, CMV, and polyomacis-acting elements (enhancer) and a selectable marker can provide aphenotypic trait for selection (e.g. dihydrofolate reductase or neomycinresistance for mammalian cells or amplicillin/tetracyclin resistance forE. coli). Selection of the appropriate vector containing the appropriatepromoter and selection marker is well within the level of those skilledin the art.

Preferably, the coding region for the construct is under the control ofan inducible promoter. In comparison to a constitutive promoter, aninducible promoter is preferable because it allows for suitable controlof the concentration of the reporter in the cell, therefore themeasurement of changes in signals are greatly facilitated.

For example, expression can be controlled using the Tet-on & Tet-offsystem Clontech (Mountain View, Calif.). Under the control of thispromoter, gene expression can be regulated in a precise, reversible andquantitative manner. Briefly, for Tet-on system, the transcription ofdownstream gene only happens when doxycycline is present in the culturemedium. After the transcription for a certain period of time, one canchange culture medium to deplete doxycycline, thus, stop the synthesisof new reporter proteins. Therefore, there is no background from newlysynthesized reporter proteins, and one may be able to see a fasterchange after toxin treatment.

Local environments for the construct other than living cells are alsocontemplated, including for example, cytosol of lysed cells, andsynthetic media that contains one or more enzymes capable of degradingthe cleavable fragment when cleaved from the reporter molecule, butincapable or much less capable of degrading the cleavable fragment priorto cleavage from reporter molecule.

It is further contemplated to provide an isolated polynucleotidemolecule encoding a construct described above. The construct ispreferably an expression vector comprising the polynucleotide moleculeoperably linked to a promoter. A preferable promoter is an induciblepromoter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B schematically depict an exemplary construct and methodof the inventive concept. FIG. 1A depicts a construct of the inventiveconcept. FIG. 1B depicts a method of the inventive concept, where theconstruct is cleaved by a botulinum toxin.

FIGS. 2A, 2B, and 2C depict results obtained from testing of differentreporting constructs. FIG. 2A shows photomicrographs of transformedcells and transformed cells following exposure to BoNT/A. FIG. 2Bgraphically depicts results of fluorescence emissions measurements fromtransformed cells exposed to BoNT/A and control transformed cells. FIG.2C shows results of SDS-PAGE followed by immunoblotting and probing withanti-SNAP-25 of the products of exposure of various constructs toBoNT/A.

FIG. 3 graphically depicts the results of fluorescence measurements fromtransformed cells expressing a reporting construct of the inventiveconcept. Results from the same set of cells are shown for theBioSentinel method (based upon characterization of YFP degradation) onthe left, and for the prior art method based on FRET emission (Loss ofFRET) from the construct on the right.

DETAILED DESCRIPTION

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing figures in which like numerals represent like components.

FIGS. 1A and 1B diagram a construct and an exemplary assay in whichBoNT/A cell-based reporters are used to detect BoNT/A activity by lossof YFP fluorescence.

FIG. 1A schematically depicts BioSentinel's BoCell™ A BoNT/A construct.The reporter fluorophore, YFP, and the normalization fluorophore, CFP,are coupled by a cleavage sequence, SNAP-25 (green). SNAP-25palmitoylation localizes the reporter to a plasma membrane.

FIG. 1B shows detection of BoNT/A activity by loss of YFP fluorescence.The YFP moiety is directly excited leading to fluorescence emission inthe absence of BoNT/A. Cleavage of the reporter by BoNT/A releases aC-terminal reporter fragment containing the YFP moiety into the cytosol.The fragment is rapidly degraded and, thus, YFP emission is lost. TheCFP signal is still used to control for cell-to-cell reporter expressionlevels and cell density.

Surprisingly, not all fluorescent proteins related to YFP are effectiveas the reporter fluorophore. For example, FIGS. 2A, 2B, and 2C provideevidence that reporters containing YFP or the closely related derivativeVenus can detect BoNT/A activity in cells, but not mCherry ormStrawberry. Here, Neuro2A cells were grown in a 96-well plate to 70%confluency and transiently transfected using Lipofectamine 2000(Invitrogen™), with reporters containing the indicated N-terminal andC-terminal (N-term/C-term) fluorophore pairs. After 24 h, cells wereincubated in the presence or absence of 10 nM BoNT/A at 37° C. for 72 hin 100 μl of phenol red-free MEM medium.

FIG. 2A shows BoNT/A-induced changes in fluorescence responses.Semi-automated YFP and CFP fluorescence measurements were performedusing a Nikon™ TE2000-U fluorescent microscope with 20× magnificationand Nikon NIS Elements 3.4 software. Shown are randomly selected fieldspseudo-colored for the C-terminal/N-terminal fluorescent protein (FP)fluorescence ratio. Ratios were calculated from emissions collected upondirect excitement of each fluorophore.

FIG. 2B represents fluorescence ratios and BoNT/A sensitivities of thecell-based reporters. 30 randomly selected cells per condition wereanalyzed for fluorescence ratios in the presence or absence of 10 nMBoNT/A. The average signal from the 30 cells from 5 microscopic fieldson 3 different wells is shown. Cells exhibiting over-saturatedfluorescence were excluded.

FIG. 2C shows a blot showing that BoNT/A was active in cells regardlessof the reporter. All reporters show some cleavage in the presence ofBoNT/A, and all native SNAP25s are cleaved. Cells were transfected andtreated with BoNT/A as described above but scaled up into 6-well plates.After 72 h incubation with BoNT/A, cells were washed 3× with serum-freeMEM, collected by scraping, and lysed using M-Per Lysis Buffer(Pierce™). 40 μg of cell lysate was subjected to SDS-PAGE beforetransfer to nitrocellulose paper and immunoblot analysis using anantibody directed against SNAP-25 (clone 71.2, Synaptic Systems). Arrowsindicate the position of the full-length (closed) and cleaved (open)forms of the reporters. Full-length (*) and cleaved (**) native SNAP-25are indicated.

Viewed from another perspective, the inventive subject matter can beextended beyond cleavable substrates, to any assay having a constructwith a reporter that can be de-protected, and then degraded in somemanner by the cytosol or other local environment. For example, asusceptible reporter could be modified to include a ‘bait’ domain thatis used to screen against a library of recombinant proteins that couldpossibly bind with the bait domain. Without the bait domain protected bya binding protein, the susceptible reporter will be degraded. In such anassay, cells expressing binding proteins will form a complex to protectthe susceptible reporter from degradation, while cells expressing abinding partner to the bait will light up. The bait domain couldadvantageously be a small peptide, and the binding partners could bemembers of a library of proteins (or protein mutants). The system couldalso be reversed such that there is a library of bait domains testedagainst a single test protein (or test protein library).

In each of these instances it is considered advantageous to include asecond reporter that is not degraded post-exposure by the cytosol orother local environment, or is at least degraded much more slowlypost-exposure than the first reporter.

Still further, whereas the reporter can conveniently be selected fromsuitable fluorophores, it is contemplated that the reporter could bereplaced or augmented by any other protein or other component with adefined function that is known to (a) have a relatively fast turnover inthe cell without protection, and (b) that can be protected byinteraction with a binding partner. Defined functions includetranscription activators for reporter gene, repressors for lethal genes,etc (anything that can be easily identified or selected against).

FIG. 3 shows an embodiment in whichata was collected for both YFPdegradation and loss of FRET according to the state of the art practicefrom the exact same plates of cells. For YFP degradation, directly andsingularly excited YFP emissions (top, Ex500, Em526) and CFP emissions(middle, Ex434, Em470) are collected. Those emission are then backgroundsubtracted and the YFP emission is divided by CFP emission to controlfor cell density and reporter expression in the individual wells. Thatemission ratio (YFP/CFP, bottom) is then used for the essay report.

For loss of FRET, FRET emissions (top, Ex434, Em526) and CFP emissions(middle, Ex434, Em470) are collected. Those emissions are thenbackground subtracted, and the FRET emission is divided by CFP emissionto control for cell density and reporter expression in the individualwells. That emission ratio (FRET/CFP, bottom) is shown here to compareto the normal method.

The key comparison is the loss of directly excited YFP versus the lossof FRET emission. From the comparison between the measurements and thecorresponding curves, it becomes immediately apparent that the overalldynamic range for YFP degradation is much larger than the dynamic rangeof loss of FRET emissions. In some cases, there is no difference,statistically, between cells treated with no BoNT versus sells treatedwith saturating concentrations of BoNT when looking solely at the rawFRET emissions. For the loss of FRET method, the BoNT dose response onlybecomes clear after dividing the FRET emission by the CFP (donor)emission. The CFP (donor) emission shows a small increase emission dueto de-quenching in response to reporter cleavage.

In summary, the loss of FRET method reports BoNT-induced changes in thereporter very poorly, or not at all, and therefore cannot be thereforeused for a correct qualitative and quantitative determination. Incontrast, preferred methods contemplated herein have a high degree ofspecificity and reproducibility, which allow one to rely on the data forboth the qualitative and quantitative analysis.

Genetic Construction of Alternative Reporters

Twenty alternate fluorophore constructs were generated in four plasmidbackgrounds. Below is the internal name of each construct with a briefdescription of the background and cloned fragment. A more detailedsummary of the construction methods follows.

Alternative Construct Plasmid Reporter Name Background DescriptionMechanism pMD0076a pcDNA4/TO mRaspberry-SNAP-YFP 1 pMD0076b pcDNA4/TOmCherry-SNAP-YFP 1 pMD0077 pIRES SNAP-YFP, CFP 3 pMD0078 pECFP-C1Synapsin promoter, 2 (Modified) SNAP-YFP pMD0079 pcDNA4/TO SNAP-YFP 2pMD0080 pECFP-C1 SNAP-YFP 2 (Modified) pMD0081 pECFP-C1mRaspberry-SNAP-YFP 1 (Modified) pMD0082 pECFP-C1 mCherry-SNAP-YFP 1(Modified) pMD0090 pIRES SNAP-Venus, CFP 3 pMD0091 pcDNA4/TO SNAP-Venus2 pMD0092 pECFP-C1 SNAP-Venus 2 (Modified) pMD0097 pcDNA4/TOmKate2-SNAP-YFP 1 pMD0098 pcDNA4/TO TagRFP-SNAP-YFP 1 pMD0099 pECFP-C1mKate2-SNAP-YFP 1 (Modified) pMD0100 pECFP-C1 TagRFP-SNAP-YFP 1(Modified) pMD0103 pBudCE4.1 SNAP-YFP, CFP 3 pMD0104 pBudCE4.1 SNAP-YFP,mRaspberry 3 pMD0105 pBudCE4.1 SNAP-YFP, mCherry 3 pMD0106 pBudCE4.1SNAP-Venus, CFP 3 pMD0107 pBudCE4.1 SNAP-Venus, mRaspberry 3 pMD0108pBudCE4.1 SNAP-Venus, mCherry 3pMD0076a, pMD0076b, pMD0097, and pMD0098

Constructs were generated by amplifying the mRaspberry, mCherry, mKate2,and TagRFP fluorophores with engineered KpnI and XhoI restriction sites.The amplified fragments and the previously used pcDNA4/TO BoCell vectorgenerated were then digested with KpnI/XhoI. The vector DNA, minus theexcised CFP, was then ligated with the mRaspberry, mCherry, mKate2, andTagRFP fragments to create the final vectors.

pMD0079 and pMD0091

For pMD0079, SNAP YFP was amplified with engineered BamHI and XhoIrestriction sites. The amplified fragment and pcDNA4/TO vector DNA werethen digested with BamHI/XhoI and then ligated together. pMD0091 wasthen generated by amplifying the Venus fluorophore with engineered EcoRIand XbaI restriction sites. The amplified Venus fragment and pMD0079were then digested with EcoRI/XbaI. The pMD0079 vector DNA, minus theexcised YFP, was then ligated with the Venus fragment to create pMD0091.

pMD0077 and pMD0090

For pMD0077, SNAP YFP was amplified with engineered NheI and XhoIrestriction sites. The amplified fragment and ORES vector DNA were thendigested with NheI/XhoI and then ligated together. The CFP fluorophorewas then amplified with engineered XbaI/NotI restriction sites. Theamplified fragment and previously generated SNAP YFP-containing pIRESvector were then digested with XbaI/NotI and ligated together to createpMD0077. pMD0090 was then generated by amplifying the Venus fluorophorewith engineered EcoRI and MluI restriction sites. The amplified Venusfragment and pMD0077 were then digested with EcoRI/MluI. The pMD0077vector DNA, minus the excised YFP, was then ligated with the Venusfragment to create pMD0090.

pMD0078 and pMD0080

For pMD0080, SNAP YFP was amplified with engineered NheI and XhoIrestriction sites. Then amplified fragment and pECFP-C1 were thendigested with NheI/XhoI. The vector DNA, minus the excised CFP, was thenligated with the SNAP YFP to create pMD0080. pMD0078 was then generatedby amplifying the synapsin promoter with engineered AseI and NheIrestriction sites. The amplified fragment and pMD0080 were then digestedwith AseI/NheI. The pMD0080 vector DNA, minus the excised CMV promoter,was then ligated with the synapsin promoter to create pMD0078.

pMD0081, pMD0082, pMD0099, pMD0100

Constructs were generated by amplifying the mRaspberry, mCherry, mKate2,and TagRFP fluorophores with engineered NheI and XhoI restriction sites.The amplified fragments and original BoCell construct from Min (pECFP-C1background) were then digested with NheI/XhoI. The BoCell construct,minus the excised CFP fragment, was then ligated with the mRaspberry,mCherry, mKate2, and TagRFP fragments to create pMD0081, pMD0082,pMD0099, and pMD0100.

pMD0092

For pMD0092, the Venus fluorophore was amplified with engineered EcoRIand XbaI restriction sites. The amplified fragment and pMD0080 were thendigested with EcoRI/XbaI. The pMD0080, minus the excised YFP fragment,was then ligated with the Venus fragment to generate pMD0092.

pMD0103, pMD0104, and pMD0105

The SNAP YFP construct was amplified with engineered XbaI and BamHIrestriction sites. The amplified fragment and pBudCE4.1 vector were thendigested with XbaI/BamHI and ligated together. The CFP, mRaspberry, andmCherry fluorophores were then amplified with engineered KpnI and BglIIrestriction sites. The amplified fragments and previously generatedpBudCE4.1 vector containing SNAP YFP were then digested with KpnI/BglIIand ligated together to generate pMD0103, pMD0104, and pMD0105.

pMD0106, pMD0107, and pMD0108

The SNAP Venus construct was amplified with engineered XbaI and BamHIrestriction sites. The amplified fragment and pBudCE4.1 vector were thendigested with XbaI/BamHI and ligated together. The CFP, mRaspberry, andmCherry fluorophores were then amplified with engineered KpnI and BglIIrestriction sites. The amplified fragments and previously generatedpBudCE4.1 vector containing SNAP Venus were then digested withKpnI/BglII and ligated together to generate pMD0106, pMD0107, andpMD0108.

Primary Screening of the Alternative Reporters

Neuro2A cells were seeded into 96-well plates and allowed to expand24-48 h before transiently transfecting the cells using the abovegenetic constructs and Lipofectamine 2000™ according to themanufacturer's instructions. Transfected cells were allowed to recoverfor 24 hr before applying 0 or 30 nM BoNT/A holotoxin and incubating thecells an additional 24 hr at 37° C., 5% CO₂. Cells were then imagedusing a Nikon-TE2000U fluorescence microscope taking a minimum of threeimages per condition. Fluorescence emissions were collected usingfilters appropriate for the listed fluorophores. Total fluorescenceemissions were also collected using a Varioskan™ fluorescence microplatereader using appropriate excitation and emission wavelength settings.

Fluorescence microscopy data was processed to gate out over expressing(saturated) cells based on pixel intensities for a given channel. Totalemissions from each channel were then collected and, when indicated, theBoNT/A-responsive YFP or Venus emissions were divided by theBoNT/A-unresponsive CFP, RFP (mKate2, mRaspberry, or mCherry), orexogenously added membrane dye (Alternative Reporter 2) emissions.

Each reporter construct, using the data collected above, was analyzedfor the following: Cellular targeting of each reporter was judged by thepresence of uniform fluorescence expression on the plasma membrane. Poorplasma membrane targeting was associated with the presence of bright,punctate spots within the cell. Reporters lacking plasma membranetargeting were eliminated from further consideration. Total fluorescenceemissions and, thus total reporter expression, were judged by theemissions of a given fluorescent probe relative to background emissions.Probes that did not give a signal >2 times that of background wereeliminated from further consideration.

Secondary Screening of the Alternative Reporters: BoNT/A-dose Responses

Genetic constructs that passed primary screening were transientlytransfected into cells as described above but using varying DNAconcentrations. Varying the DNA concentration generated cells withvarying levels of reporter expression. After transfection and a 24 hrrecovery period, the transfected cells were titrated with 10 pM to 30 nMBoNT/A allowed to further incubate. After incubation, fluorescenceemissions were collected using a Varioskan fluorescence microplatereader using appropriate excitation and emission wavelength settings.For all experiments, test reporter responses were directly compared tothe current BoCell reporter CFP-SNAP25-YFP that was transientlytransfected in parallel.

Fluorescence emissions for the BoNT/A-response YFP were divided by theBoNT/A-unresponsive CFP, RFP (mKate2, mRaspberry, or mCherry), orexogenously added membrane dye (Alternative Reporter 2) emissionsgenerating an emission ratio. The emission ratio was then plotted as afunction of BoNT/A concentration. Data was compared to the BoCellreporter. Test reporter suitability was qualitatively assessed bycomparison to the BoCell reporter: Does BoNT/A elicit a similar responsewith the test reporter compared to the current BoCell reporter?

Secondary Screening of the Alternative Reporters: FRET Emissions

Reporter genetic constructs were transfected into cells plated on glasscover slips using Lipofectamine according to the manufacturer'sprotocol. For each construct, single fluorophore controls were alsotransfected. After a 24 hr recovery period, cells were treated with orwithout 30 nM BoNT. Using the controls for each reporter, images werecaptured by fluorescence microscopy and the images used to calibrateFRET determinations by a three-filter set method. Reporters were thenevaluated for FRET efficiency in the presence and absence of BoNT/A. Forall experiments, test reporter responses were directly compared to thecurrent BoCell reporter CFP-SNAP25-YFP that was transiently transfectedin parallel. Each test reporter was evaluated for the presence of FRETand whether FRET emissions were responsive to BoNT/A. The conclusion wasthat FRET emissions do not represent a reliable screening method, inline with the conditions already observed in example 3.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. An artificial construct comprising: a nucleicacid sequence encoding a hybrid protein comprising (a) areporter-containing portion comprising a first fluorophore, wherein thereporter-containing portion is coupled to (b) a cleavage site thatinteracts with a botulinum toxin in a manner that cleaves thereporter-containing portion from a remainder of the hybrid protein,wherein the cleavage site comprises at least a portion of a motifselected from the group consisting of SNAP-25, synaptobrevin, andsyntaxin, and (c) a second portion comprising a control fluorophore,wherein the cleavage site is interposed between the reporter containingportion and the second portion, wherein the reporter-containing portionand the second portion are positioned in the hybrid protein such thatFRET emission from the first fluorophore does not show a decreasingtrend relative to increasing concentration of the botulinum toxin. 2.The artificial construct of claim 1, wherein the reporter-containingportion comprises at least one fluorophore selected from the groupconsisting of Yellow Fluorescent Protein (YFP), Citrine, Venus, and aYPet protein.
 3. The artificial construct of claim 1, wherein thecontrol fluorophore comprises at least one fluorophore selected from agroup consisting of CFP, mStrawberry, and mCherry.
 4. A hybrid protein,comprising: (a) a reporter-containing portion comprising a firstfluorophore, wherein the reporter-containing portion is coupled to (b) acleavage site that interacts with a botulinum toxin in a manner thatcleaves the reporter-containing portion from a remainder of the hybridprotein, wherein the cleavage site comprises at least a portion of amotif selected from the group consisting of SNAP-25, synaptobrevin, andsyntaxin, and (c) a second portion comprising a control fluorophore,wherein the cleavage site is interposed between the reporter containingportion and the second portion, wherein the reporter-containing portionand the second portion are positioned in the hybrid protein such thatFRET emission from the first fluorophore does not show a decreasingtrend relative to increasing concentration of the botulinum toxin, andwherein the second portion is selected and positioned within the hybridprotein in a manner such that, an increased concentration of botulinumtoxin does not lead to a coincidental increase in fluorescent emissionof the control fluorophore.
 5. The hybrid protein of claim 4, whereinthe reporter-containing portion comprises at least one fluorophoreselected from the group consisting of Yellow-Fluorescent Protein (YFP),Citrine, Venus, and a YPet protein.
 6. The hybrid protein of claim 4,wherein the control fluorophore comprises at least one fluorophoreselected from a group consisting of CFP, mStrawberry, and mCherry.
 7. Amodified cell, comprising: a hybrid protein, the hybrid proteincomprising (a) a reporter-containing portion comprising a firstfluorophore, wherein the reporter-containing portion is coupled to (b) acleavage site that interacts with a botulinum toxin in a manner thatcleaves the reporter-containing portion from a remainder of the hybridprotein, wherein the cleavage site comprises at least a portion of amotif selected from the group consisting of SNAP-25, synaptobrevin, andsyntaxin, and (c) a second portion comprising a control fluorophore,wherein the cleavage site is interposed between the reporter containingportion and the second portion, wherein the reporter-containing portionand the second portion are positioned in the hybrid protein such thatFRET emission from the first fluorophore does not show a decreasingtrend relative to increasing concentration of the botulinum toxin, andwherein the second portion is selected and positioned within the hybridprotein in a manner such that, an increased concentration of botulinumtoxin does not lead to a coincidental increase in fluorescent emissionof the control fluorophore.
 8. The modified cell of claim 7, wherein thecell is derived from a cell line selected from the group consisting of aneuronal cell, a neuroendocrine tumor cell, a hybrid cell, and a stemcell.
 9. The modified cell of claim 7, wherein the reporter-containingportion comprises at least one fluorophore selected from the groupconsisting of Yellow Fluorescent Protein (YFP), Citrine, Venus, and aYPet protein.
 10. The modified cell of claim 7, wherein the controlfluorophore comprises at least one fluorophore selected from a groupconsisting of CFP, mStrawberry, and mCherry.
 11. A kit comprising: amodified cell, the modified cell comprising a a hybrid protein, thehybrid protein comprising (a) a reporter-containing portion comprising afirst fluorophore, wherein the reporter-containing portion is coupled to(b) a cleavage site that interacts with a botulinum toxin in a mannerthat cleaves the reporter-containing portion from a remainder of thehybrid protein, wherein the cleavage site comprises at least a portionof a motif selected from the group consisting of SNAP-25, synaptobrevin,and syntaxin, and (c) a second portion comprising a control fluorophore,wherein the cleavage site is interposed between the reporter containingportion and the second portion, wherein the reporter-containing portionand the second portion are positioned in the hybrid protein such thatFRET emission from the first fluorophore does not show a decreasingtrend relative to increasing concentration of the botulinum toxin, andwherein the second portion is selected and positioned within the hybridprotein in a manner such that, an increased concentration of botulinumtoxin does not lead to a coincidental increase in fluorescent emissionof the control fluorophore.
 12. The kit of claim 11, wherein themodified cell is derived from a cell line selected from the groupconsisting of a neuronal cell, a neuroendocrine tumor cell, a hybridcell, and a stem cell.
 13. The kit of claim 11, further comprising acell culture medium.